Effect Of EX Vivo Expanded Regulatory T Cells Induced By Donor Dendritic Cells On Recipient Tumor Immunity

ObjectiveThis study performed to test whether adoptive transfer of ex vivo expanded Tregs would inhibit anti-tumor immunity of recipients, and to coculture Tregs with donor antigen after expansion would induce antigen specificity of Tregs.Methods1. Mature donor dendritic cells were generated through inducing bone marrow cells of C57BL/6 (H-2b) or BALB/c (H-2d) mice in the presence of recombinant murine GM-CSF, IL-4 and TNF-α.2. CD4+CD25+ Tregs were sorted by MACS from C57BL/6 or BALB/c mice, and some Tregs were expanded ex vivo with recombinant murine IL-2 (100U/ml) and anti-CD3/CD28 microbeads for 14 days.3. Expanded BALB/c Tregs were cocultured with C57BL/6 mature dendritic cells or expanded C57BL/6 Tregs were cocultured with BALB/c mature dendritic cells in the presence of lower concentration of recombinant murine IL-2 (200U/ml) for 7 days.4. Purity of mature dendritic cells and Tregs were tested by flow cytometry, in vitro inhibition of CD4+CD25" T cells response by Tregs was measured by CFSE dilution mixed lymphocyte reaction test.5. We used a skin allograft model to evaluate the effects of Tregs.6. C57BL/6 and BALB/c mice were inoculated with B16-F10 murine melanoma cell (H-2b) via tail vein to establish tumor models.7. C57BL/6 and BALB/c mice were inoculated with expanded or induced Tregs, and B16-F10 cells were injected intravenously after 24h.8. After 14 days, mice were sacrificed and the black tumor nodules in lungs were counted.Results1. Result showed ex vivo expanded or induced Tregs inhibited CD4+CD25T cells response, as compared to fresh isolated Tregs.2. Expanded Tregs and induced Tregs prolonged survival of MHC fully mismatched skin graft (14.5 days Vs 9 days, p<0.05).3. Two weeks after inoculated of 5×105 B16-F10 cells, there were (93±12) tumor nodules in lung of C57BL/6 mice. Adoptive transfusion of 1×107 in vitro expanded Tregs one day before inoculation of B16-F10 significantly increased tumor nodules in the lung of C57BL/6 mice harvest two weeks after inoculation (355±15), however adoptive transfusion of 1×107 induced Tregs into C57BL/6 mice one day before inoculation of 5×105 B16-F10 cells, the numbers of tumor nodules were (195±13). Compared to mice without Tregs transfusion, the difference was statistically significant (p<0.05).4. After inoculated of 5×105 B16-F10 cells, there were (9±6) tumor nodules in lung of BALB/c mice in the 14th day, and the tumor nodules were significantly increased to (124±4) in lung of BALB/c mice when adoptive transfusion of 1×107 in vitro expanded Tregs one day before inoculation of B16-F10, when injected 1×107 induced Tregs into BALB/c mice on day-1, and 5×105 B16-F10 cells were injected intravenously after 24h, we found there were (297±18) tumor nodules in lung of BALB/c mice in the 14th day, Compared to mice without Tregs transfusion, the difference was statistically significant (p<0.05).Conclusions1. The purity of fresh Tregs was over than 95%by MACS.2. We can get plenty of Tregs with immunosuppressive function by ex vivo amplification.3. Ex vivo expanded Tregs were able to suppress tumor immunity of recipient to MHC matched (recipient derived) or mismatched tumor (donor derived).4. Coculture of these expanded Tregs with donor dentric cells could induce antigen specificity.